complement c3 rabbit pab  (Proteintech)

Journal: Frontiers in Immunology

Article Title: EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies

doi: 10.3389/fimmu.2018.02910

Figure Lengend Snippet: EF-Tu is present at the NTHi cell surface. (A) Rabbit antibodies raised against recombinant NTHi EF-Tu recognize different NTHi clinical isolates. NTHi ( n = 6) were analyzed by flow cytometry. Mean values from three independent experiments are shown and error bars represent standard error of the mean (SEM). (B) Representative flow cytometry profiles of NTHi 3655 and KR334. (C) Localization of EF-Tu in NTHi 3655 and KR334 was analyzed by transmission electron microscopy (TEM). (D) Encapsulated H. influenzae represented by H. influenzae type b (MinnA and Eagan) as compared to the non-encapsulated mutant H. influenzae RM804 based upon Hib Eagan. (E) Representative flow cytometry profiles of Hib Eagan and the capsule mutant Hib RM804. (F) Surface concentration of EF-Tu on Hib Eagan and RM804 as measured by TEM. Anti-EF-Tu IgG was used in all experiments. Antibodies were affinity purified from rabbits that had been immunized with recombinant NTHi EF-Tu produced in E. coli . Flow cytometry analyses were performed using H. influenzae incubated with rabbit anti-EF-Tu IgG followed by secondary FITC-conjugated goat anti-IgG pAbs. Background represents bacteria incubated with only the secondary antibody, and was defined as < 2% of positive bacterial cells. Error bars indicate SEM. For TEM visualization, a gold-labeled secondary antibody was used.

Article Snippet: The cells were thereafter stained with FITC-conjugated goat anti-rabbit C3 pAbs (MP Biomedicals, Santa Ana, CA, United States), followed by analysis using a FACSverse TM flow cytometer.

Techniques: Recombinant, Flow Cytometry, Transmission Assay, Electron Microscopy, Mutagenesis, Concentration Assay, Affinity Purification, Produced, Incubation, Labeling

Journal: Frontiers in Immunology

Article Title: EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies

doi: 10.3389/fimmu.2018.02910

Figure Lengend Snippet: Four main immunoreactive epitopes are detected at the surface of EF-Tu. (A) Map of the synthetic peptides covering the EF-Tu protein sequence that were used for epitope mapping. (B) The reactivity of anti-EF-Tu pAbs from rabbits ( n = 4) immunized with recombinant EF-Tu, and of sera from rabbits immunized with whole NTHi (3655 or 334) ( n = 5) were tested against synthetic EF-Tu peptides using a dot blot (Supplementary Figure ). Mean values obtained by scanning densitometry are shown. (C) The surface-exposed peptides ID 3, 9, 12, and 15 are indicated on the model of the EF-Tu crystal structure (Protein Data Bank entry 1dg1). These peptides corresponded to EF-Tu 41−65 , EF-Tu 161−185 , EF-Tu 221−245 , and EF-Tu 281−305 . Peptides ID 9, 12, and 15 were located within the CNBr-generated fragments indicated in Figure . (D) Specific antibodies against peptides ID 3, 9, 12, and 15 recognize EF-Tu on the surface of NTHi 3655, as revealed by flow cytometry. Mean values from 9 experiments are shown. The anti-peptide antibodies were affinity-purified from sera obtained from rabbits immunized with full-length recombinant EF-Tu. Error bars indicate SEM. Representative flow cytometry results are shown for anti-peptide Abs directed against peptide ID 3 and 12. Flow cytometry analysis was performed using NTHi 3655 cells incubated with the anti-EF-Tu IgG or peptide-specific antibodies followed by secondary FITC-conjugated pAbs. Background represents bacteria incubated with secondary antibody only.

Article Snippet: The cells were thereafter stained with FITC-conjugated goat anti-rabbit C3 pAbs (MP Biomedicals, Santa Ana, CA, United States), followed by analysis using a FACSverse TM flow cytometer.

Techniques: Sequencing, Recombinant, Dot Blot, Generated, Flow Cytometry, Affinity Purification, Incubation

Journal: Frontiers in Immunology

Article Title: EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies

doi: 10.3389/fimmu.2018.02910

Figure Lengend Snippet: Antibodies directed against EF-Tu are functional and induce bactericidal activity via complement activation. (A) C3 deposition was assessed in the presence of rabbit antiserum against full-length EF-Tu. NTHi strains KR334 and 3655 were incubated with EF-Tu antiserum, and baby rabbit complement was used as a complement source. Flow cytometry analysis was performed using a specific anti-C3 antibody. Pre-immune serum was used as a negative control. Mean values from 6 experiments are shown. A representative flow cytometry profile is also shown. Background represents bacteria incubated with the secondary antibody only. (B) Serum bactericidal activity (SBA) was performed to assess NTHi killing induced by anti-EF-Tu antibodies. Rabbit antibodies directed against full-length EF-Tu or peptide ID 3, 9, and 12 significantly induced the classical pathway of complement activation in the presence of baby rabbit complement. Bacterial killing represents differences between incubation with active or heat-inactivated baby rabbit complement. Control represents bacterial killing from baby rabbit complement with the non-related anti-OprG pAb detecting a Pseudomonas aeruginosa outer membrane protein. Mean values from 5 separate experiments are indicated. Statistical significances were calculated using the Mann-Whitney test. Error bars indicate SEM.

Article Snippet: The cells were thereafter stained with FITC-conjugated goat anti-rabbit C3 pAbs (MP Biomedicals, Santa Ana, CA, United States), followed by analysis using a FACSverse TM flow cytometer.

Techniques: Functional Assay, Activity Assay, Activation Assay, Incubation, Flow Cytometry, Negative Control, MANN-WHITNEY